JP2016206648A - Laser scan microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To shorten time required for acquiring image data in comparison with when sequentially implementing an in-focus action to acquire image data on a broad area in laser scan microscope devices.SOLUTION: A laser scan microscope device 100 includes: an irradiation unit 1 that converges laser light to irradiate a sample 6 with the laser light; an optical detection unit 3 that detects light to be emitted from an irradiation position; XY scanning means that scans the laser light in an X-direction and Y-direction; and Z scanning means that scans the laser light in a Z-direction, and acquires information about the sample 6. Upon acquiring XY two-dimensional image data through scanning of the irradiation position in the X-direction and Y-direction by use of the XY scan means, by scanning the irradiation position even in the Z-direction by means of the Z scanning means, the laser scan microscope device 100 is configured to detect the light through scanning the irradiation position even in the Z-direction by use of the Z scanning means, and thereby acquire XY two-dimensional image data having information on light emitted from the irradiation position different in a Z coordinate stored, respectively and including at least two pixels.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a laser scanning microscope apparatus.

  When observing a sample using a microscope, the width of the visual field that can be observed at a time is limited by the width of the visual field of the objective lens. Therefore, when observing a sample larger than the field of view of the objective lens, the entire sample cannot be observed at a time.

  On the other hand, in pathological diagnosis, there is a desire to observe the entire sample in order to prevent oversight of the observation target. Therefore, in pathological diagnosis, before observing a specific region of interest in detail using a high-magnification objective lens, a relatively wide region of the sample is observed using a low-magnification objective lens. Decisions are made.

  As a method of acquiring an image of a region wider than one field of view of the microscope, a plurality of images are acquired by moving the field of view in a direction (X direction, Y direction) perpendicular to the optical axis direction (Z direction) of the microscope, A method for obtaining a single large image by combining a plurality of acquired images is known. However, the position in the Z direction where the sample exists (Z coordinate) may be different for each field of view, such as when the sample has undulations. When the field of view is moved in the X and Y directions, the focal plane of the microscope and the sample are not aligned. It is possible that it will shift.

  Therefore, Patent Literature 1 describes a microscope system that performs a focusing operation every time the visual field is moved in the X direction and the Y direction, and acquires a wide-area image that is in focus on the whole.

Japanese Patent Laid-Open No. 9-281405

  In Patent Document 1, a series of operations for acquiring an image in a visual field after adjusting a focal position to a sample by a focusing operation is repeated every time the visual field is moved to acquire a wide area image. Therefore, there is a problem that it takes time to acquire a wide area image.

  Accordingly, in view of the above-described problems, the present invention provides a series of operations for acquiring an image within a visual field after performing a focusing operation in the time required for acquiring image data in a laser scanning microscope apparatus. The purpose is to shorten the time compared with the case of acquiring wide-area image data repeatedly.

  A laser scanning microscope apparatus according to one aspect of the present invention includes an irradiation unit that condenses laser light by an objective lens and irradiates the sample, and light emitted from the irradiation position of the laser light that is condensed and irradiated. The light detection unit to detect, and the laser beam irradiated to the sample by the irradiation unit scans in the X direction perpendicular to the optical axis direction of the objective lens and the Y direction perpendicular to the optical axis direction and the X direction. XY scanning means, comprising: Z scanning means for scanning the laser light irradiated onto the sample by the irradiation unit in a Z direction parallel to an optical axis direction of the objective lens The light is detected while the irradiation position is scanned in the X direction and the Y direction by the XY scanning means, and the detected light information is the X coordinate and Y of the irradiation position. When acquiring the XY two-dimensional image data stored for each pixel corresponding to the target, the Z scanning means scans the irradiation position in the Z direction to detect the light, thereby irradiating with different Z coordinates. XY two-dimensional image data including at least two pixels each storing information of the light emitted from a position is acquired.

  According to the present invention, in the laser scanning microscope apparatus, the time required for acquiring an image is repeated for each time the visual field moves by repeating a series of operations for acquiring an image within the visual field after performing a focusing operation. This can be shortened compared to the case of acquiring image data.

It is a figure which shows typically the structure of the laser scanning microscope apparatus which concerns on 1st Embodiment. It is a flowchart which shows the operation | movement in the 2nd measurement mode of the laser scanning microscope apparatus which concerns on 1st Embodiment. It is a figure which shows typically the time-dependent change of the irradiation position of the spot which concerns on 1st Embodiment, and the timing of data taking-in. It is a figure which shows typically the time-dependent change of the irradiation position of the spot which concerns on 2nd Embodiment, and the timing of data acquisition. (A) The figure which showed typically the change of the position of the measurement point which concerns on 1st Embodiment, (b) The figure which showed typically the change of the position of the measurement point which concerns on 2nd Embodiment. . It is a figure which shows typically the time-dependent change of the irradiation position of the spot which concerns on 3rd Embodiment, and the timing of data acquisition. It is a flowchart which shows the operation | movement in the 2nd measurement mode of the laser scanning microscope apparatus which concerns on 4th Embodiment. It is the figure which looked at the locus | trajectory of the irradiation position of the spot which concerns on 4th Embodiment from the X direction. It is a figure which shows typically the structure of the laser scanning microscope apparatus which concerns on 5th Embodiment. It is a figure which shows typically the structure of the laser scanning microscope apparatus which concerns on 6th Embodiment.

  Hereinafter, several embodiments according to the laser scanning microscope apparatus of the present invention will be described. In addition, although these embodiment is an example of the best embodiment in this invention, this invention is not limited only to the various structure and numerical value which are demonstrated in these embodiment. These configurations and numerical values can be appropriately selected.

(First embodiment)
(Device configuration)
First, the configuration of a laser scanning microscope apparatus 100 (hereinafter referred to as “apparatus 100”) according to the first embodiment will be described with reference to FIG. FIG. 1 is a diagram schematically showing the configuration of the apparatus 100.

  The apparatus 100 irradiates at least a part of the inside of the sample 6 or the surface of the sample 6 with a laser beam as a spot. Or the like) is detected. Then, the laser light detects the light L by two-dimensionally scanning the irradiation position S of the sample 6. The apparatus 100 thereby acquires the two-dimensional distribution information of the light L and acquires the two-dimensional image data of the sample 6. When detecting the light L, the light L, which is light emitted from the irradiation position S, may be directly detected, or reflected light, transmitted light, scattered light, etc. of the laser light irradiated on the sample 6 may be detected. The light L may be detected from the intensity change.

  The type of the apparatus 100 is not particularly limited as long as the apparatus 100 is a laser scanning microscope apparatus that collects and irradiates laser light as a spot on the sample 6 and scans the irradiation position S with the inside of the sample 6 or the surface of the sample 6. Specifically, a nonlinear optical microscope apparatus, a confocal laser microscope apparatus, etc. are mentioned. The nonlinear optical microscope apparatus is a microscope apparatus using a nonlinear optical effect, and examples of the nonlinear optical effect include multiphoton excitation, multiphoton absorption, stimulated Raman scattering (SRS), and coherent anti-Stokes Raman scattering (CARS). Anti-Stokes Raman Scattering), Coherent Stokes Raman Scattering (CSRS), Second Harmonic Generation (SHG), and the like.

  As illustrated in FIG. 1, the apparatus 100 includes an irradiation unit 1, a sample stage 2, a light detection unit 3, a control unit 4, a data acquisition unit 5, and a data processing unit 8.

  The irradiation unit 1 includes a light source 11, an optical scanner 12, and a first objective lens 13. The irradiation unit 1 is a part that condenses the laser light emitted from the light source 11 by the first objective lens 13 and irradiates the inside of the sample 6 or the surface of the sample 6 as a spot.

  The light source 11 is a light source that emits laser light. The type of laser light emitted from the light source 11 is not particularly limited, and its wavelength band, output, pulse light / CW light classification, and the like can be arbitrarily selected. In addition, although this embodiment demonstrates the apparatus 100 using only one light source, it is good also as a structure which has a some light source. For example, the apparatus 100 may have a configuration of an SRS microscope apparatus that uses the first laser and the second laser at the same time. Alternatively, the apparatus 100 may be a laser scanning fluorescence microscope apparatus having a plurality of lasers respectively corresponding to excitation wavelengths of a plurality of fluorescent dyes in the sample as the light source 11. Further, the apparatus 100 may be a fluorescence microscope apparatus that has a wavelength tunable light source as the light source 11, and an operator selects a wavelength as necessary to selectively excite a fluorescent dye in a sample. When the light source 11 includes a plurality of light sources, the light source 11 may further include a multiplexing unit that combines the laser beams from the plurality of light sources 11 of the irradiation unit 1.

  The optical scanner 12 emits the laser beam emitted from the light source 11, collected by the first objective lens 13 and irradiated as a spot to the sample 6 with respect to the optical axis O of the first objective lens 13. This is a portion that scans in a vertical plane direction (hereinafter referred to as “XY direction”). That is, the optical scanner 12 is an X scanning unit that scans the spot irradiation position in the X direction perpendicular to the optical axis O. The optical scanner 12 is also Y scanning means for scanning the spot irradiation position in the Y direction perpendicular to the optical axis O and perpendicular to the X direction. That is, the optical scanner 12 is an XY scanning unit that scans the irradiation position of the spot in the X direction and the Y direction.

  The first objective lens 13 condenses the laser light emitted from the light source 11 and irradiates the sample 6 as a spot.

  The sample stage 2 is a portion on which the sample 6 is placed. The sample 6 is placed between the first objective lens 13 and the second objective lens 31 (described later) by being placed on the sample stage 2 by the operator. The sample stage 2 can be moved in a direction parallel to the optical axis O (hereinafter referred to as “Z direction”) or an XY direction by the control unit 4. Thereby, the relative position with respect to the apparatus 100 of the sample 6 mounted on the sample stage 2 can be moved, and the scanning area | region of the laser beam scanned by the irradiation part 1 can be moved.

  The light detection unit 3 detects light emitted from the irradiation position S inside the sample 6 or the surface of the sample 6 of the laser light irradiated as a spot by the irradiation unit 1. Thereby, the light detection part 3 can acquire the information regarding the sample 6 from the irradiation position of a spot. The light detection unit includes a second objective lens 31, an optical filter 32, and a detector 33.

  The second objective lens 31 collects light including light emitted from the irradiation position of a spot in or on the sample 6 such as fluorescence emitted from the sample 6 or laser light transmitted through the sample 6. A condenser lens may be used as the second objective lens.

  The optical filter 32 is a part that extracts light having a required wavelength by blocking light having a predetermined wavelength out of the light collected by the second objective lens 31. For example, when detecting fluorescence as the light L, the optical filter 32 blocks light corresponding to the wavelength of light that excites the phosphor in the sample 6 and transmits only light having the wavelength of the fluorescence to be detected. In addition, when detecting stimulated Raman scattering light as the light L, the optical filter 32 blocks light of one wavelength among the two types of light irradiated to the sample 6 and allows only light of the other wavelength to pass.

  The extraction of light having a necessary wavelength may be performed by another method instead of the optical filter 32. For example, instead of the optical filter 32, a spectroscope such as a dichroic mirror or a diffraction grating may be used.

  The detector 33 is a part that detects light transmitted through the optical filter 32. The detector 33 receives the light that has passed through the optical filter 32, performs photoelectric conversion, and outputs an electrical signal. At this time, the detector 33 outputs the light intensity of the received light as the voltage signal intensity. As the detector 33, for example, a photomultiplier tube (photomultiplier tube, PMT) can be used. The detector 33 may be configured by combining a photodiode and a lock-in amplifier.

  The control unit 4 is a part that controls the optical scanner 12 and the sample stage 2. The control unit 4 includes a sample stage control unit 41 and an optical scanner control unit 42.

  The sample stage control unit 41 controls driving of a motor (not shown) that moves the sample stage 2 so that the sample stage 2 moves in the X direction, the Y direction, and the Z direction, respectively.

  The movement of the sample stage 2 whose driving is controlled by the sample stage control unit 41 is roughly divided into an XY movement and a Z movement.

  The XY movement is movement of the sample stage 2 in the X direction and the Y direction. Thereby, the scanning region (hereinafter referred to as “field of view”) of the spot scanned by the irradiation unit 1 moves in the XY direction. Although the width of the field of view is limited by the first objective lens 13 and the optical scanner 12, by moving the field of view in the XY direction by XY movement in this way, observation in a wider range than one field of view can be performed.

  In this embodiment, the optical scanner 12 scans the spot in the X direction and the Y direction, and the sample stage control unit 41 performs the XY movement of the field of view. However, the present invention is not limited to this. That is, the sample stage 2 may be configured to scan the spot in the X direction and the Y direction. When the sample stage 2 is configured to scan the spot in the X direction and the Y direction, the Z scanning means also serves as the XY scanning means. However, since scanning with the optical scanner 12 can be performed at a higher speed, a configuration in which the optical scanner 12 scans the spot in the X direction and the Y direction is preferable.

  Z movement is movement of the sample stage 2 in the Z direction. Thereby, the position in the Z direction of the spot irradiated by the irradiation part 1 moves. By moving the position of the spot in the Z direction, observation can be performed while changing the depth of the sample 6 in the visual field. The apparatus 100 according to the present embodiment can scan a spot in the Z direction by the sample stage control unit 41 in the same manner as the spot is scanned in the X direction and the Y direction by the optical scanner 12. That is, in the present embodiment, the sample stage control unit 41 and the sample stage 2 are Z scanning means. In the present embodiment, the sample stage control unit 41 moves the sample stage 2 in the Z direction to move the position of the spot in the Z direction. However, the present invention is not limited to this. For example, the first objective lens 13 may be configured to be movable in the Z direction, and the position of the spot in the Z direction may be moved by moving the first objective lens 13 in the Z direction. That is, the irradiation unit 1 may be a Z scanning unit. For example, the relative positional relationship between the first objective lens 13 and the sample 6 can be changed by connecting a piezo element, tuning fork, motor, and gear to the first objective lens 13.

  The data acquisition unit 5 associates the signal intensity of the electrical signal received from the light detection unit 3 with the X and Y coordinates of the spot position calculated from the control amounts of the optical scanner 12 and the sample stage 2 in an XY two-dimensional manner. This is the part that captures image data. The data capturing unit 5 may capture XYZ three-dimensional image data in which the Z coordinate of the spot position is further associated with the information.

  The apparatus 100 is a transmission type laser scanning microscope apparatus, but may be a reflection type laser scanning microscope apparatus. In that case, the 1st objective lens 13 is implement | achieved by the structure which serves as the role of the 2nd objective lens 31 as an example.

  The data processing unit 8 is a part that receives data transmitted from the data fetching unit 5 and processes the received data. For example, the data processing unit 8 takes in image data associated with different fields of view, and generates wide area image data by pasting them. In addition, the data processing unit 8 can perform data processing of various data such as XY two-dimensional image data by a method such as multivariate analysis or machine learning, and can visualize components included in the sample 6. The data processing unit 8 may include a storage unit (not shown) for temporarily storing various types of data, storing data processing results, various conditions for measurement, and the like.

  In addition, the apparatus 100 may include an image display unit 71 and an instruction input unit 72.

  The image display unit 71 is connected to the data processing unit 8 and displays a measurement result, an image generated by the data processing unit 8, various conditions at the time of measurement, and the like. As the image display unit 71, for example, a flat panel display or the like can be used.

  The instruction input unit 72 is a part that is connected to the data capturing unit 5 via the data processing unit 8, and is used to input various conditions and the like at the time of measurement by an operator who uses the apparatus 100.

(First measurement mode)
Next, the operation of the apparatus 100 when acquiring XY two-dimensional image data of one field of view at a specific Z coordinate will be described. In this specification, this operation is referred to as a “first measurement mode”.

  Laser light is emitted from the light source 11, introduced into the optical scanner 12, and scanned in the XY directions by the optical scanner 12 so that the spot position when the light is condensed by the first objective lens 13 is scanned in the XY directions. Is done. The laser light that has passed through the optical scanner 12 is condensed by the first objective lens 13 and irradiated as a spot on the inside of the sample 6 or the surface of the sample 6.

  The apparatus 100 (light detection unit 3) detects fluorescence emitted from the sample 6 and transmitted through the sample 6 as the light L when excited by the laser beam. Therefore, the fluorescence emitted from the inside of the sample 6 or the spot on the surface of the sample 6 is collected by the second objective lens 31. At this time, the apparatus 100 collects not only the fluorescence emitted from the sample 6 but also the laser light (excitation light) transmitted through the sample 6 by the second objective lens 31. In addition, although this embodiment demonstrates the apparatus 100 which detects fluorescence as the light L, the light L is not limited to this. The light L may be light generated by nonlinear optical effects such as multiphoton excitation, multiphoton absorption, stimulated Raman scattering, coherent anti-Stokes Raman scattering, coherent Stokes Raman scattering, and second harmonic generation.

  The light condensed by the second objective lens 31 passes through the optical filter 32. Here, the optical filter 32 according to this embodiment has different transmission characteristics depending on the wavelength, and is configured to block light having the wavelength of the laser light (excitation light) emitted from the light source 11. Since the wavelength of the fluorescence is different from the wavelength of the laser light, the laser light (excitation light) can be blocked by passing through the optical filter 32 and only the fluorescence can be transmitted.

  The fluorescence transmitted through the optical filter 32 is guided to the detector 33. The detector 33 receives the fluorescence and outputs an electric signal having a voltage value corresponding to the intensity of the fluorescence.

  The electrical signal output from the detector 33 is subjected to analog / digital conversion (AD conversion) by the data capturing unit 5. A digital signal obtained by AD conversion is transmitted to a storage unit (not shown). In synchronization with this, the data fetching unit 5 acquires the position information of the irradiation position of the spot and the wavelength information of the excitation light and the fluorescence from the control unit 4. The data capturing unit 5 generates XY two-dimensional image data in which the fluorescence intensity is stored for each pixel corresponding to the XY coordinates based on the various information thus obtained.

  As described above, the apparatus 100 can acquire XY two-dimensional image data at a specific Z coordinate. Further, by acquiring the XY two-dimensional image data by moving the Z coordinate of the spot by the Z scanning means, a plurality of XY two-dimensional image data having different Z coordinates (depths) can be acquired.

  However, if a plurality of XY two-dimensional image data is acquired by changing the Z coordinate for each field of view, it takes time to acquire the XY two-dimensional image data. Furthermore, since the amount of data acquired is enormous, subsequent analysis takes time. For this reason, when the visual field is moved in the XY direction for wide-area observation, it is preferable that the number of XY two-dimensional image data acquired for each visual field is small. However, in order to reduce the number of XY two-dimensional image data for each field of view, for example, when XY two-dimensional image data is acquired only for a specific Z coordinate, image data for which information on the sample 6 cannot be obtained depending on the field of view is obtained. Will occur. This problem is particularly noticeable when the sample 6 has “undulation” and the Z coordinate where the sample 6 exists differs depending on the position in the XY direction. Therefore, when performing wide-area observation, the apparatus 100 acquires XY two-dimensional image data in a “second measurement mode” described later.

(Second measurement mode)
Hereinafter, the operation of the apparatus 100 in the second measurement mode will be described with reference to FIGS. 2 and 3. FIG. 2 is a flowchart showing the operation of the apparatus 100 in the second measurement mode. FIGS. 3A and 3B are diagrams schematically showing the temporal change of the irradiation position of the spot and the timing of data acquisition in the second measurement mode of the apparatus 100. FIG.

  First, the operator operates the instruction input unit 72 to move the sample stage 2 in the Z direction, and inputs parameters for scanning the spot irradiation position in the Z direction (S201). At this time, the operator inputs, as parameters, the scanning range in the Z direction of the spot irradiation position and the scanning speed or scanning frequency of the spot irradiation position to the instruction input unit 72.

  The scanning range of the spot in the Z direction is a range from the lower end to the upper end of the Z coordinate of the irradiation position of the spot, and this corresponds to the moving range of the sample stage 2 in the Z direction. The scanning range in the Z direction of the spot is preferably set so as to include at least all the Z coordinates where the sample 6 exists.

  As the sample 6, for example, a biological tissue section can be used, and the sample 6 is often a thin sample having a thickness of about several μm. The sample 6 is often placed on the sample stage 2 while being sandwiched between a slide glass (not shown) and a cover glass (not shown) and held in a liquid such as water. Therefore, the sample 6 often has “undulation”, and the Z coordinate where the sample 6 exists is often different depending on the position in the XY direction.

  As described above, the sample 6 may not be sandwiched between the slide glass (not shown) and the cover glass (not shown), and is simply placed on the slide glass (not shown). It may be placed on top. Also in this case, the Z coordinate where the sample 6 exists is often different depending on the position in the XY direction.

  When the sample 6 has “undulation” as described above, the width of the Z coordinate where the sample 6 varies depending on the position in the XY direction is generally about several μm to 10 μm in many cases. Therefore, the scanning range in the Z direction of the spot input in S201 is preferably set to about 2 to 3 times. For example, when the swell of the sample 6 is about 10 μm, it may be set to about 20 μm to 30 μm. preferable. Thereby, the scanning range of the Z direction of a spot can be set so that all the Z coordinates in which the sample 6 exists may be included. However, the value of the scanning range in the Z direction of the spot is not limited to this, and can be arbitrarily set according to the form of the sample 6.

  In the present embodiment, since the spot is scanned in the Z direction by moving the sample stage 2 in the Z direction, the scanning speed of the spot in the Z direction corresponds to the moving speed of the sample stage 2 in the Z direction. .

  Next, the apparatus 100 starts moving the sample stage 2 in the Z direction based on the parameters input by the operator, and starts scanning the spot irradiation position in the Z direction (S202). Subsequently, the apparatus 100 uses the optical scanner 12 to start XY scanning of the spot irradiation position (S203). After that, the device 100 starts data acquisition by the data acquisition unit 5 (S204).

  As described above, when the apparatus 100 acquires the XY two-dimensional image data by detecting the light L and acquiring the data while scanning the irradiation position of the spot in the X direction and the Y direction, the apparatus determines the irradiation position of the spot as Z. The light L is detected and data is captured while scanning in the direction. In the present embodiment, the scanning in the X direction and the scanning in the Y direction are periodically repeated until the XY scanning in one field of view is completed. Moreover, it is preferable that the scanning in the Z direction is repeated periodically until the XY scanning in one field of view is completed. The scanning in the Z direction is preferably performed by vibrating the sample stage 2 in the Z direction.

  As shown in FIG. 3A, the apparatus 100 scans the spot irradiation position in the Z direction by the Z scanning means while the spot irradiation position is scanned in the Y direction by the XY scanning means. Alternatively, as shown in FIG. 3B, while the spot irradiation position is scanned in the X direction by the XY scanning means, the spot irradiation position is also scanned in the Z direction by the Z scanning means. Hereinafter, the case of FIG. 3A will be described, but the same applies to the case of FIG.

  In the present embodiment, when scanning the spot irradiation position in the XY direction, scanning in the X direction is performed in the reverse direction for each line of the scanning line. The scanning is performed by a method, but is not limited thereto. For example, when taking in data, a scanning method in which scanning in the X direction is always performed in the same direction may be employed.

  In the present embodiment, as shown in FIG. 3A, the spot irradiation position is not scanned in the Z direction while the spot irradiation position is being scanned in the X direction. When the scanning in the X direction and the data acquisition for one scanning line are completed, the spot irradiation position is scanned in the Y direction. At this time, the apparatus 100 scans the spot irradiation position in the Z direction while scanning the spot irradiation position in the Y direction. That is, after the light detection unit 3 detects the light L at the spot irradiation position, the spot irradiation position is scanned in the Y direction by the XY scanning means, and the spot irradiation position is scanned in the Z direction by the Z scanning means. To do. At this time, after the spot irradiation position is moved in the Y direction, the spot irradiation position may be moved in the Z direction, or the movement in the Y direction and the movement in the Z direction are performed at the same time. The position may be moved diagonally.

  When the movement of the irradiation position of the spot is completed, the light detection unit 3 detects the light L and takes in data. Thereby, the information of the light L can be acquired at the irradiation position where the Z coordinate of the irradiation position of the spot is different from the X coordinate or the Y coordinate. The apparatus 100 repeats the above-described operation until the data acquisition for the predetermined XY region is completed (S205), so that the information on the light L emitted from the irradiation position having a different Z coordinate is obtained for each pixel in the XY plane. The stored XY two-dimensional image data can be acquired. That is, the XY two-dimensional image data acquired by the apparatus 100 is two-dimensional image data including at least two pixels each storing information on the light L emitted from the irradiation position having a different Z coordinate. In other words, in the XY two-dimensional image data constituting one image, a plurality of Z coordinate values are distributed and stored for each pixel on the XY plane. Examples of the data configuration include a configuration in which different Z coordinates are randomly stored in each pixel in the XY plane, and a configuration in which the Z coordinates regularly differ between adjacent pixels.

  Note that the operation when the apparatus 100 acquires XY two-dimensional image data in the second measurement mode is not limited to the above specific example. That is, when acquiring the XY two-dimensional image data by scanning the spot irradiation position in the X direction and the Y direction, the spot irradiation position is also set in the Z direction before the scanning in the X direction and the Y direction is completed. What is necessary is just to scan. At this time, the light L may be detected at a plurality of Z coordinates for one measurement point (pixel) in the XY plane, but the number of Z coordinates at this time is the number of Z coordinates in the entire scanning range in the Z direction. Set less than the number.

  By acquiring the XY two-dimensional image data in this way, the apparatus 100 can reduce the number of measurement points compared to the case of acquiring all the XY two-dimensional image data for each Z coordinate. Here, the “measurement point” refers to a point where data is captured by the data capturing unit 5 among the irradiation positions of the spots.

  For example, when the operator sets the scanning range in the Z direction of the spot irradiation position via the instruction input unit 72 to include 10 measurement points, from the measurement points having the same X coordinate and Y coordinate. It is assumed that the data acquisition is set to be performed for only one Z coordinate. Then, conventionally, XY two-dimensional image data has been acquired for each Z coordinate, but in the case of the apparatus 100 according to the present embodiment, the number of measurement points can be reduced to 1/10 times. That is, the apparatus 100 can thin out a signal corresponding to an XYZ three-dimensional space with respect to a Z coordinate and project the signal on an XY two-dimensional plane. As a result, according to the present embodiment, the time required to acquire the image data can be reduced to about 1/10 times.

  In this case, depending on the irradiation position of the spot, it may not be located inside the sample 6, and the light L derived from the sample 6 may not be acquired. In such a case, the apparatus 100 may complement the signal intensity at the point where the light L derived from the sample 6 can be acquired among the adjacent measurement points.

  The apparatus 100 acquires XY two-dimensional image data in one field of view as described above, so that XY two-dimensional image data roughly showing the form of the sample 6 is obtained even when the sample 6 has “swell”. Can be acquired at high speed.

  When the acquisition of XY two-dimensional image data from one field of view is completed (Yes in S205), the apparatus 100 moves the sample stage 2 in the X direction or the Y direction (S207). Thereby, the visual field is moved in the XY directions.

  The apparatus 100 repeats acquisition of XY two-dimensional image data in each field of view while repeating movement of the field of view in the X direction or Y direction. Then, the data capture unit 5 combines the acquired plurality of XY two-dimensional image data to generate wide-area XY two-dimensional image data. Accordingly, the apparatus 100 can acquire XY two-dimensional image data of an area wider than the size of one visual field.

  The method of moving the visual field in the X direction or the Y direction is not particularly limited. For example, first, the field of view may be moved in the X direction, and then after a predetermined number of fields have been moved, one field of view may be moved in the Y direction, and the field of view may be moved again in the X direction. That is, the visual field may be scanned by raster scanning. The number of visual fields in the X direction and the Y direction is preferably set so that the entire sample 6 can be accommodated. For example, you may prescribe | regulate by the size of the area | region of the acquired wide XY two-dimensional image data like 10 mm for each of a X direction and a Y direction.

  The above operation is repeated, and when the acquisition of XY two-dimensional image data is completed for all preset regions (Yes in S206), the XY scanning of the spot irradiation position by the optical scanner 12 is stopped (S208). Thereafter, the Z movement of the spot irradiation position by the sample stage 2 is stopped (S209).

  The apparatus 100 acquires XY two-dimensional image data in one field of view as described above, so that XY two-dimensional image data roughly showing the form of the sample 6 is obtained even when the sample 6 has “swell”. Can be acquired at high speed. In particular, when acquiring wide-area XY two-dimensional image data with a large number of fields of view, the effect of shortening the measurement time in each field of view is added, so that the effect of this embodiment appears more remarkably.

  As described above, the apparatus 100 acquires a plurality of XY two-dimensional image data while moving the field of view in the X direction or the Y direction, and combines them to generate wide-area XY two-dimensional image data. Thereby, even if the sample 6 has “swell”, wide-area XY two-dimensional image data that roughly shows the form of the sample 6 can be acquired at high speed. That is, the apparatus 100 according to the present embodiment can reduce the time required to acquire image data.

Even if the XY two-dimensional image data in each field of view is coarse as described above, the state and form of the sample 6 over the entire area based on the wide-area XY two-dimensional image data finally obtained. It is not so much problem to grasp the rough. For example, when an objective lens having a numerical aperture (NA) of about 1 is used, the spot size is generally 1 μm ³ or less, and therefore the interval between measurement points is usually set to 1 μm or less. The spot size depends on the wavelength and NA. When the NA is small, the spot size is large, but is about 10 μm ³ at most. Therefore, in the entire image acquired from a wide area of about 10 mm square, which is 1000 times larger than the spot size, even if there is a pixel in which data from the sample 6 is not stored, the entire form is not greatly affected. .

  As described above, the apparatus 100 according to the present embodiment can reduce the time required to acquire wide-area XY two-dimensional image data. Furthermore, since the data amount of the image data to be acquired can be reduced, the calculation cost and calculation time when analyzing the image data by a technique such as multivariate analysis can be reduced.

(Second Embodiment)
Next, an apparatus 200 according to the second embodiment will be described. Since the apparatus configuration of the apparatus 200 and the operation in the first measurement mode are the same as those of the apparatus 100, a description thereof will be omitted, and differences in the operation in the second measurement mode will be described with reference to FIGS. FIG. 4A and FIG. 4B are diagrams schematically showing the temporal change of the spot irradiation position and the data acquisition timing in the second measurement mode of the apparatus 200.

  The apparatus 100 operates so that the spot irradiation position is scanned in the Z direction by the Z scanning unit while the spot irradiation position is scanned in either the X direction or the Y direction by the XY scanning unit. However, in the apparatus 200, the spot irradiation position is scanned while the spot irradiation position is scanned in the X direction by the XY scanning means and while the spot irradiation position is scanned in the Y direction by the XY scanning means. Are scanned in the Z direction by the Z scanning means.

  In other words, in the first embodiment, after the scanning of the spot irradiation position in the X direction or the Y direction is completed, the scanning of the spot irradiation position in the Z direction, the Y direction, or the X direction is performed. It was. However, in the present embodiment, the spot irradiation position is also scanned in the Z direction without waiting for the scanning of the spot irradiation position in the X direction or the Y direction to be completed.

  FIG. 4A schematically shows an example of the change with time of the irradiation position of the spot and the timing of data acquisition in the second measurement mode of the apparatus 200. That is, the apparatus 200 also scans in the Z direction while scanning the spot irradiation position in the X direction. The apparatus 200 also performs scanning in the Z direction while scanning the spot irradiation position in the Y direction.

  FIG. 5A shows that, in the apparatus 100, scanning in the Z direction is not performed while scanning the spot irradiation position in the X direction, and Z scanning is performed while scanning in the Y direction is performed. It is the figure which showed typically the change of the position of the measurement point at the time of scanning to. 5A and 5B are views seen from the X direction. Although not depicted in FIG. 5A, at each measurement point (black circle), the irradiation position of the spot is scanned in the back direction of the paper surface or the front side of the paper surface, that is, in the X direction.

  On the other hand, FIG. 5B shows a change in the position of the measurement point when scanning is performed in the Z direction while scanning the spot irradiation position in both the X direction and the Y direction. FIG. In addition, the solid line arrow of FIG.5 (b) represents the locus | trajectory of the scanning to the X direction of the irradiation position of a spot.

  In both the case of FIG. 5A and the case of FIG. 5B, the state where the measurement point exists inside the sample 6 and the state where it does not exist are repeated. However, in the case of FIG. 5B (second embodiment, apparatus 200), the measurement point is located inside the sample 6 as compared to the case of FIG. 5A (first embodiment, apparatus 100). The frequency of repeating the existing state and the non-existing state is high. That is, the apparatus 200 scans the spot irradiation position in the Z direction more frequently than the apparatus 100. Thus, although depending on the type and form of the sample 6, information in the Z direction is averaged more uniformly than the apparatus 200, and XY two-dimensional image data representing the form of the sample 6 can be obtained more densely. it can.

  FIG. 4B schematically shows another example of the change with time of the irradiation position of the spot and the timing of data acquisition in the second measurement mode of the apparatus 200. In other words, the apparatus 200 scans in the Y direction while scanning the spot irradiation position in the X direction, and simultaneously scans in the Z direction. In this case, it is possible to reduce the time required to acquire wide-area XY two-dimensional image data, compared to the case of FIG.

(Third embodiment)
Next, an apparatus 300 according to the third embodiment will be described. Since the apparatus configuration of the apparatus 300 and the operation in the first measurement mode are the same as those of the apparatus 100 (first embodiment), a description thereof will be omitted, and differences in the operation in the second measurement mode will be described with reference to FIG. . FIG. 6 is a diagram schematically showing the temporal change of the spot irradiation position and the data acquisition timing in the second measurement mode of the apparatus 300.

  In the first embodiment and the second embodiment, the scanning frequency in the Z direction of the spot irradiation position is set to be equal to or lower than the scanning frequency in the X direction or the Y direction. However, in the third embodiment, the scanning frequency in the Z direction of the spot irradiation position is set to be higher than the scanning frequency in the X direction or the Y direction.

  FIG. 6 schematically shows an example of the temporal change of the spot irradiation position and the data acquisition timing in the second measurement mode of the apparatus 300. Here, as in the first embodiment, the irradiation position of the spot is scanned in the X direction and also in the Z direction, and when scanning in the Y direction, no scanning is performed in the Z direction. Similar to the first and second embodiments, the third embodiment can reduce the time required to acquire wide-area XY two-dimensional image data.

  Here, a case is shown in which the scanning frequency in the Z direction of the spot irradiation position is set to four times the scanning frequency in the X direction. Thus, the frequency of scanning in the Z direction can be increased by making the frequency of scanning in the Z direction of the spot irradiation position higher than the frequency of scanning in the X or Y direction. Thereby, the frequency which repeats the state in which the measurement point exists in the sample 6 and the state which does not exist can be raised. As a result, the information in the Z direction is averaged more uniformly, and XY two-dimensional image data that more closely represents the form of the sample 6 can be acquired.

  Here, the case where the scanning frequency in the Z direction at the spot irradiation position is set to be higher than the scanning frequency in the X direction or the Y direction in the first embodiment has been described. Even if it is applied to the form, the same effect can be obtained.

(Fourth embodiment)
Next, an apparatus 400 according to the fourth embodiment will be described. Since the apparatus configuration of the apparatus 400 and the operation in the first measurement mode are the same as those in the first embodiment, a description thereof will be omitted. Differences in the operation in the second measurement mode will be described with reference to FIGS. FIG. 7 is a flowchart showing the operation of the apparatus 400 in the second measurement mode. FIG. 8 is a view of the locus of the spot irradiation position in this embodiment as viewed from the X direction.

  The apparatus 400 scans the spot irradiation position in the Z direction once (from the start point 81 to the end point 82), and then determines the position of the sample 6 in the Z direction. Based on this, the apparatus 400 changes the start point 81 or the end point 82 of the spot when the next scanning in the Z direction of the spot irradiation position is performed. Thereby, even if the “swell” of the sample 6 is large, the measurement can be performed following the “swell” of the sample 6. Hereinafter, an example of this feedback control will be described with reference to the flowchart of FIG.

  FIG. 7 shows a part of the flowchart (S204) of FIG. 2 in detail. The operation of the apparatus 400 is the same as the flowchart of FIG. 2 except for S204.

  After scanning of the spot irradiation position in the Z direction (S202) and scanning in the XY direction (S203) are started, the apparatus 400 captures data while scanning the spot irradiation position in the Z direction (S2041). ). The irradiation position of the spot is scanned back and forth in the Z direction, but the above-described scanning and data capturing operation is continued until the scanning in the Z direction is completed for one scanning line 80. When the spot irradiation position reaches the end point 82 and one scan in the Z direction is completed (Yes in S2042), the apparatus 400 determines the position of the sample 6 in the Z direction based on the data acquired by the scan line 80. (S2043). Based on the position of the sample 6 obtained in S <b> 2043, the apparatus 400 sets the Z-direction scanning start point 81 in the Z direction so that the entire sample 6 can be scanned with the spot in the Z direction on the next scanning line 80. Moving.

  For example, when determining the position of the sample 6 in the Z direction (S2043), based on the Z coordinates of a plurality of measurement points from which signals having values exceeding a predetermined threshold value are obtained, their center Z coordinates Zc. Can be calculated as the position of the sample 6. Then, as shown in FIG. 8, the position (Zc) of the sample 6 in the Z direction coincides with the Z coordinate of the center of the next scanning line in the Z direction (the Z coordinate of the center of the start point 81 and the end point 82). The starting point 81 of scanning in the Z direction is moved in the Z direction (S2044).

  Thereby, even if the “swell” of the sample 6 is large, the measurement can be performed following the “swell” of the sample 6. Further, according to the present embodiment, the width of Z scanning can be set smaller than in the first to third embodiments. Thereby, the case where the irradiation position of a spot is not located in the sample 6 or on the sample 6 can be reduced, and measurement points where data derived from the sample 6 cannot be obtained can be reduced. As a result, XY two-dimensional image data that more closely represents the form of the sample 6 can be acquired.

  In the present embodiment, the starting point 81 of scanning in the Z direction is moved in the Z direction so that Zc is the position in the Z direction of the sample 6 and the Z coordinate of the center of the scanning line in the next Z direction coincides with Zc. However, it is not limited to this. For example, the existence range in the Z direction of the sample 6 is acquired based on the Z coordinates of a plurality of measurement points from which a signal having a value exceeding a predetermined threshold is obtained, and the center Z of the scanning line in the next Z direction is acquired. The starting point 81 of scanning in the Z direction may be moved in the Z direction so that the coordinates are within the acquired range.

(Fifth embodiment)
Next, a laser scanning microscope apparatus 101 (hereinafter referred to as “apparatus 101”) according to a fifth embodiment will be described.

  First, the device configuration of the device 101 will be described with reference to FIG. FIG. 9 is a diagram schematically illustrating the configuration of the apparatus 101. The apparatus 101 can change the wavelength of light irradiated on the sample 6 by the light source unit 1.

  In the fifth embodiment, measurement is performed while changing the wavelength of light irradiated on the sample 6. According to the fifth embodiment, in the unlikely event that the constituent component included in the sample 6 is unknown, the wavelength of the light irradiated to the sample 6 in order to obtain the light L derived from the constituent component of the sample 6 Even if the appropriate wavelength is unknown, the distribution information of the constituent components contained in the sample 6 can be acquired. Incidentally, the constituents contained in the sample 6 are rarely unknown to the operator. Further, as will be described later, the apparatus 101 can acquire spectrum data at each irradiation position by performing measurement while changing the wavelength of the light irradiated on the sample 6. The apparatus 101 can also acquire detailed information regarding the constituent components contained in the sample 6 by analyzing the spectrum data.

  The apparatus configuration of the apparatus 101 is the same as the apparatus configuration of the apparatus 100 except for the configuration of the light source unit 1 and the control unit 4, and therefore only the configuration of the light source unit 1 and the control unit 4 will be described.

  The light source unit 1 included in the apparatus 101 includes a first light source 111, a second light source 112, a wavelength changing unit 113, and a multiplexing unit 114.

  The first light source 111 and the second light source 112 are light sources that emit a first laser beam and a second laser beam, respectively. The type of laser light emitted from the first light source 111 and the second light source 112 is not particularly limited.

  The wavelength changing unit 113 is a unit that changes the wavelength of the second laser light emitted from the second light source 112. In the present embodiment, the second light source 112 is a broadband light source for generating light in which light of a plurality of wavelengths is mixed. The wavelength changing unit 113 is a spectroscopic unit, which splits the second laser light emitted from the second light source and emits only light having an arbitrary wavelength. The wavelength changing unit 113 according to the present embodiment can sweep the wavelength of the emitted light at a constant period. As the wavelength changing unit 113, for example, a galvano scanner, a resonant scanner, an acousto-optic variable filter, a polyhedral mirror type filter, or the like can be used.

  The combining unit 114 is a unit that combines the first laser light emitted from the first light source and the second laser light having the wavelength changed from the wavelength changing unit.

  In the present embodiment, the light source unit 1 has two light sources, the first light source 111 and the second light source 112. However, the present invention is not limited to this, and one light source may be used. There may be three or more. In the present embodiment, the wavelength changing unit 113 changes the wavelength of the second laser light. However, the present invention is not limited to this, and the wavelength of the first laser light may be changed. The wavelength of the laser beam and the wavelength of the second laser beam may be changed.

  The control unit 4 included in the apparatus 101 includes a sample stage control unit 41, an optical scanner control unit 42, and a wavelength changing unit control unit 43.

  The apparatus 101 changes the difference (wavelength difference) between the wavelength of the first laser light and the wavelength of the second laser light by changing the wavelength of the second laser light by the wavelength changing unit 113. Can do. Hereinafter, although the case where the apparatus 101 changes a wavelength difference is demonstrated, it is not limited to this, The apparatus 101 has a light source which radiate | emits the laser beam of a single wavelength, and the wavelength of the laser beam radiate | emitted from the light source The same applies to the case of changing.

  By using near-infrared light as the first laser light and the second laser light, nonlinear Raman scattered light such as stimulated Raman scattered light or coherent anti-Stokes Raman scattered light is suitably detected as light L. be able to.

(First measurement mode)
The apparatus 101 in the first measurement mode changes the wavelength difference by the wavelength changing unit 113 at each spot irradiation position and detects the light L and captures data n times. The apparatus 101 scans the irradiation position of the spot in the X direction and the Y direction by the XY scanning unit while detecting the light L and taking in the data at each irradiation position. Thereby, the apparatus 101 acquires spectrum data at each irradiation position. Alternatively, the apparatus 101 may change the wavelength difference at the timing when image data for one field of view is formed by performing X scanning and Y scanning of the spot irradiation position with a single wavelength difference. In this case, the apparatus 101 changes the wavelength difference every time XY scanning is completed, and repeats XY scanning n times.

  By these operations, the apparatus 101 in the first measurement mode acquires spectrum data that stores the intensity of the light L for each of the n types of wavelength differences at each irradiation position. As a result, the apparatus 101 can acquire “spectral image data” in which spectral data at each irradiation position is stored in each pixel corresponding to the XY coordinates of each irradiation position.

  Furthermore, the apparatus 101 can acquire the distribution information of the constituent components contained in the sample 6 by analyzing the obtained spectral image data by the data processing unit 8. The analysis in the data processing unit 8 can be performed using a technique such as multivariate analysis or machine learning.

(Second measurement mode)
On the other hand, the apparatus 101 in the second measurement mode has p type (where n> p ≧ 1) wavelength differences among the n types of wavelength differences used by the apparatus 101 in the first measurement mode at each measurement point. To measure. As a result, the amount of spectral data obtained at each irradiation position can be reduced by a factor of p / n.

  The apparatus 101 in the second measurement mode detects the light L while acquiring the XY two-dimensional image data while scanning the spot irradiation position in the X direction and the Y direction, as in the other embodiments. The light L is detected by scanning the irradiation position also in the Z direction. As a result, the time required to acquire the image data can be shortened as in the other embodiments. The apparatus 101 in the second measurement mode further scans the spot irradiation position in the X direction, the Y direction, and the Z direction as described above, and changes the wavelength difference of the light applied to the sample 6 by the wavelength changing unit 113. . That is, while acquiring XY two-dimensional image data within one field of view, the apparatus 101 changes the wavelength difference of the irradiated light while scanning the spot irradiation position in the X and Z directions. Alternatively, the apparatus 101 changes the wavelength difference of the irradiated light while scanning the irradiation position of the spot in the Y direction and the Z direction while acquiring XY two-dimensional image data within one field of view. Alternatively, the apparatus 101 changes the wavelength difference of the irradiated light while scanning the irradiation position of the spot in the X direction, the Y direction, and the Z direction while acquiring XY two-dimensional image data within one field of view.

  The apparatus 101 in the second measurement mode changes the p-type wavelength difference used for measurement at each measurement point at least once, detects the light L and captures data, and acquires XY two-dimensional image data. When changing the p-type wavelength difference, all of the p-type wavelength differences may be changed, or a part of the p-type wavelength difference may be changed. That is, the apparatus 101 changes the combination of the p types of wavelength differences used by the apparatus 101 in the second measurement mode for measurement of each measurement point among the n types of wavelength differences used by the apparatus 101 in the first measurement mode. As a result, the apparatus 101 acquires XY two-dimensional image data in which information on the light L generated when light having different wavelength differences is irradiated to irradiation positions having different Z coordinates is stored for each pixel in the XY plane. be able to. In other words, in the XY two-dimensional image data constituting one image, a plurality of Z coordinate values and a plurality of wavelength difference values are distributed and stored for each pixel on the XY plane. Data configuration examples include a configuration in which different Z coordinates and different wavelength difference values are randomly stored in each pixel in the XY plane, and the Z coordinate and wavelength difference values are regularly between adjacent pixels. Different configurations are included.

  The number of times and timing of changing the wavelength difference are not particularly limited. For example, when changing the wavelength difference of the irradiated light while scanning the spot irradiation position in the X and Z directions, the spot irradiation position is changed in the X and Z directions at least once in one field of view. The p-type wavelength difference used for measurement at each measurement point may be changed while scanning. For example, if p = 1, measurement is performed using one type of wavelength difference at each irradiation position of the spot, and the wavelength difference is calculated while scanning the irradiation position of the spot once in the X and Z directions. You may change (n-1) times. The wavelength changing unit 113 preferably changes the wavelength difference periodically.

  By performing measurement while changing the wavelength difference of light used for measurement as described above, it is possible to perform measurement within one field of view, compared to the case where measurement is performed using the same p types of wavelength differences at all measurement points within one field of view. It is possible to increase the types of wavelength differences used when acquiring XY two-dimensional image data. According to the fifth embodiment, the number of types of wavelength differences used for measurement can be made larger than p types when viewed in the entire field of view. Therefore, there is a higher possibility that a signal from the component included in the sample 6 can be obtained than when measurement is performed using the same p types of wavelength differences for all measurement points in one field of view. Therefore, in the unlikely event that the constituent component contained in the sample 6 is unknown, an appropriate wavelength as the wavelength of the light irradiated to the sample 6 to obtain the light L derived from the constituent component of the sample 6 is unknown. Even so, the distribution information contained in the sample 6 can be acquired.

  According to the present embodiment, the amount of spectrum data obtained at each irradiation position can be reduced by a factor of p / n. Similarly, the data amount of the obtained spectral image data is p / p as compared to the case where measurement is performed with n types of wavelength differences at each measurement point in the second measurement mode of the first to fourth embodiments. It can be reduced n times. As an example, in the first measurement mode, the apparatus 101 changes the wavelength difference between the first laser light and the second laser light 90 times and irradiates the sample 6 to detect the stimulated Raman scattering light as the light L. Consider the case of a stimulated Raman scattering microscope that captures data 91 times. In this case, for example, if p = 1, the amount of spectral data obtained at each irradiation position can be reduced to 1/91 times, and the amount of spectral image data can also be reduced to 1/91 times. it can.

  Furthermore, according to the present embodiment, the time required to acquire the spectrum data at each measurement point can be shortened to p / n times. As a result, the time required to acquire the spectral image data can be shortened. That is, in the case of the above-described conditions, the time required to acquire the spectrum data at each measurement point is reduced to 1/91 times compared to the case where measurement is performed with n types of wavelength differences at each measurement point. be able to. Further, according to the present embodiment, the amount of spectral image data can be reduced, so that the time required for analyzing the spectral image data and generating image data indicating the distribution of the constituent components can also be shortened. .

  As described above, according to the present embodiment, XY two-dimensional acquired is obtained as compared with the case where measurement is performed with n types of wavelength differences at each measurement point in the second measurement mode of the first to fourth embodiments. The amount of data can be reduced while maintaining the quality of the image data.

  The fifth embodiment is particularly effective when performing wide-area observation on the sample 6. In the fifth embodiment, a pixel from which the light L derived from the component included in the sample 6 is obtained and a pixel from which the light L cannot be obtained coexist in one visual field. However, if it is viewed in a wide area image, it does not matter so much to roughly grasp the state and form of the sample 6. Therefore, the present embodiment is effective because the amount of data can be reduced while maintaining the image quality necessary for wide-area observation.

(Sixth embodiment)
Next, a laser scanning microscope apparatus 102 (hereinafter referred to as “apparatus 102”) according to a sixth embodiment will be described.

  The apparatus 102 is a laser scanning microscope apparatus that detects reflected light as the light L generated from the sample 6. First, the device configuration of the device 102 will be described with reference to FIG. FIG. 10 is a diagram schematically illustrating the configuration of the device 102.

  The apparatus configuration of the apparatus 102 is substantially the same as the apparatus configuration and functions of the laser microscope apparatuses of the first to fifth embodiments except for the configuration of the light detection unit 3. Next, the configuration of the light detection unit 3 will be described.

  Similarly to the above-described embodiment, the apparatus 102 irradiates at least a part of the surface of the sample 6 or the surface of the sample 6 with a laser beam, and emits light generated by fluorescence or a nonlinear optical phenomenon emitted from the laser beam irradiation position. L is detected. At this time, the light L is emitted in four directions starting from the generation source, and a part of the light L is transmitted through the sample 6, but a part of the light L is also emitted to the upper part of the sample 6 as scattered light. Of the light L, the scattered light emitted perpendicularly to the sample surface is mixed with the reflected light of the light incident on the sample 6. Therefore, in the reflection type arrangement of the light detection unit 3 in this embodiment, the light L is detected by detecting scattered light or reflected light emitted on the upper portion of the sample 6 and measuring the intensity change thereof. The reflective arrangement has the advantage that even a thick sample that cannot transmit scattered light can be measured.

  As shown in FIG. 10, the light detection unit 3 in this embodiment includes a beam splitter 14, an objective lens 13, an optical filter 32, and a detector 33. Among these, the objective lens 13 and the beam splitter 14 constitute a part of the irradiation unit 1.

  The laser light emitted from the light source 11 passes through the optical scanner 12 and the beam splitter 14, is condensed by the objective lens 13, and is irradiated onto the sample 6. The light reflected from the sample is collected by the objective lens 13, a part of the light is reflected by the beam splitter 14, and enters the optical filter 32. The optical filter 32 transmits only light having the same wavelength as the wavelength of the first light, and the transmitted light enters the photodetector 33. Note that the positions of the beam splitter 14 and the optical scanner 12 can be interchanged. Further, the light source 11 may have a plurality of light sources, similar to the light source 11 of the apparatus 101.

  Here, the beam splitter 14 may be a polarization beam splitter. In this case, the polarization of the laser beam from the light source 11 and the polarization transmission direction of the beam splitter are matched, so that the laser beam from the light source 11 is transmitted. Then, of the scattered light emitted from the sample 6, a component that does not coincide with the polarization direction of the laser light of the light source 11 is reflected by the beam splitter 14, enters the optical filter 32, and further enters the photodetector 33. Here, when the light source 11 includes a plurality of light sources, the optical filter 32 transmits only light having the same wavelength as that of the first light.

  In order to detect the light L, the scattered light from the sample 6 may be directly detected. In that case, a collecting optical system for collecting scattered light is provided separately from the objective lens 13 for incident light. The light collected by the collection optical system is incident on the optical filter 32, and the light transmitted through the optical filter 32 is incident on the photodetector 33.

  In order to arrange the light detection unit 3 more compactly and detect scattered light efficiently, it is possible to adopt a configuration in which no collection optical system is provided. In this case, an optical filter and a photodetecting element that constitutes a photodetector may be installed close to the sample so that scattered light is directly incident on the optical filter. For example, an optical filter and a light detection element are stacked and formed outside the objective lens 13 near the opening of the objective lens 13. Alternatively, an optical filter or a light detection element may be disposed inside the objective lens.

  Further, a part of the optical path between the light source 11 and the objective lens 13 or between the objective lens 13 (or the collection optical system) and the photodetector 33 may be formed of an optical fiber. At this time, a moving mechanism may be provided on the objective lens 13 side instead of the sample stage to move the laser scanning region.

1 Irradiation unit 12 Optical scanner (XY scanning means)
3 Photodetector 41 Sample stage controller (Z scanning means)
42 Optical scanner controller (XY scanning means)
5 Data acquisition part 6 Sample

Claims (13)

An irradiating unit for condensing laser light by an objective lens and irradiating the sample;
A light detection unit that detects light emitted from the irradiation position of the laser light that is collected and irradiated;
XY scanning means for scanning the laser beam irradiated onto the sample by the irradiation unit in the X direction perpendicular to the optical axis direction of the objective lens and the Y direction perpendicular to the optical axis direction and the X direction. A laser scanning microscope device,
Z scanning means for scanning the laser beam irradiated to the sample by the irradiation unit in a Z direction parallel to the optical axis direction of the objective lens,
The XY scanning means detects the light while scanning the irradiation position in the X direction and the Y direction, and information on the detected light is stored for each pixel corresponding to the X coordinate and the Y coordinate of the irradiation position. When the obtained XY two-dimensional image data is acquired, the light is detected from the irradiation position with different Z coordinates by detecting the light while scanning the irradiation position in the Z direction by the Z scanning means. A laser scanning microscope apparatus characterized in that XY two-dimensional image data including at least two pixels each storing information is acquired.   The XY scanning unit scans the irradiation position in the X direction and the Y direction, and the Z scanning unit scans the irradiation position in the Z direction to detect the light. Item 2. The laser scanning microscope apparatus according to Item 1. Further comprising wavelength changing means for changing the wavelength of the laser beam;
While the irradiation position is scanned in at least one of the X direction, the Y direction, and the Z direction, the wavelength changing unit detects the light while changing the wavelength of the laser light. 3. The laser scanning microscope apparatus according to claim 1, wherein XY two-dimensional image data including at least two pixels each storing information on the light having different wavelengths of the laser light is acquired. 4. .   The laser scanning microscope apparatus according to claim 3, wherein the wavelength changing unit periodically changes the wavelength of the laser light. The XY scanning means is means for periodically scanning the irradiation position in the X direction and the Y direction;
The laser scanning microscope apparatus according to any one of claims 1 to 4, wherein the Z scanning unit is a unit that periodically scans the irradiation position in the Z direction.   6. The cycle in which the XY scanning unit scans the irradiation position in the X direction and the Y direction is larger than a cycle in which the Z scanning unit scans the irradiation position in the Z direction. The laser scanning microscope apparatus described. The irradiation unit is
A first light source that emits a first laser beam;
A second light source that emits a second laser beam;
A combining means for combining the first laser light and the second laser light into the laser light,
The laser scanning microscope according to any one of claims 3 to 6, wherein the wavelength changing unit changes a difference between the wavelength of the first laser light and the wavelength of the second laser light. apparatus.   The laser scanning microscope apparatus according to any one of claims 1 to 7, wherein the light is light including stimulated Raman scattered light or coherent anti-Stokes Raman scattered light. The Z scanning means is based on the intensity of the light detected by scanning the irradiation position in the Z direction by the Z scanning means,
The laser scanning microscope apparatus according to any one of claims 1 to 8, wherein a Z coordinate of a start point of scanning of the irradiation position in the Z direction is changed.   The Z scanning unit is a unit that periodically scans the irradiation position in the Z direction by vibrating the sample in the Z direction. The laser scanning microscope apparatus according to the item.   4. The XY two-dimensional image data is spectral image data in which the intensity of the light with respect to the wavelength of the laser light is stored is spectral image data stored for each of the pixels. The laser scanning microscope apparatus according to any one of claims 10 to 10.   The laser scanning microscope apparatus according to claim 11, further comprising a data processing unit that processes the spectral image data by multivariate analysis.   The light detected by the light detection unit is at least one selected from transmitted light, reflected light, and scattered light emitted from the irradiation position. The laser scanning microscope apparatus according to the item.

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